The heart has evolved an adaptive response to stress, termed late preconditioning (PC), that confers powerful and sustained protection against ischemia/reperfusion injury. Previous work by us and others has shown that late PC is mediated by the upregulation of a cluster of stress-responsive proteins, namely, iNOS, HO-1, and ecSOD, which mitigate tissue injury via the combined regulation of the myocardial levels of NO and CO. Our preliminary data indicate that these proteins are organized hierarchically and interact with one another to effect cytoprotection, thereby forming a functional module. The central theme of this Program Project is to elucidate the molecular mechanisms responsible for the powerful cardioprotective effects of the iNOS-HO-1-ecSOD module, and to explore the feasibility of upregulating this module with genetic or pharmacologic approaches in order to achieve chronic prophylactic cardioprotection. The Program will consist of four Projects and four Cores. Project 1 (Bolli) will determine whether gene therapy strategies that increase myocardial NO and CO levels (iNOS, HO-1, or ecSOD gene therapy) result in a chronically-protected phenotype and will elucidate how these three-stress responsive genes interact in modulating NO and CO levels. This Project will test the novel idea that gene therapy can be used to achieve a permanent preconditioned-like state. Project 2 (Prabhu) will determine whether the same three genes (iNOS, HO-1, and ecSOD) mitigate chronic post-infarction myocardial remodeling and the resulting heart failure, and will investigate the mechanisms for these beneficial effects. The same gene therapy strategies tested in Project 1 in the setting of acute ischemia/reperfusion injury will be tested in Project 2 in the setting of post-infarction LV remodeling. This Project will test the novel idea that the iNOS-HO-1-ecSOD module is a critical protective mechanism against heart failure. Project 3 (Xuan) will investigate the signaling and molecular mechanisms Whereby NO and CO induce delayed protection, focusing on the remarkable ability of these molecules to suppress apoptosis. The central hypothesis of this Project is that the NO-CO axis plays a critical role in the cardiac adaptation to stress by virtue of its ability to genetically reprogram the heart in a manner that promotes cell survival. Project 4 (Bhatnagar) will test the hypothesis that in the preconditioned heart iNOS and HO-1 inhibit mitochondrial respiration, which decreases free radical generation, inhibits mitochondrial permeability transition, and prevents cell death. These four projects will be supported by four Cores that will provide services in mouse surgery, cardiac gene-transfer, transgenic and knockout mouse generation, viral vector production, and pathology. This Program Project is the natural evolution of our previous work on late PC - having identified NO and CO as key mediators of this adaptation, we will investigate the mechanism of protection and the therapeutic utility of the NO-CO axis. Because of the ubiquitous role of NO and CO, the results will have broad pathophysiological implications for numerous biological processes in which NO and CO are known to serve and important regulatory function. In addition, the results may provide a framework for translating cardioprotective therapies to the clinical arena.